Project Summary Kinesin and myosin are so-called `motor proteins' that can use two `feet' to walk along microtubule and actin filaments (respectively) that make up the cytoskeleton. These motors support many vital functions with the cell, including pulling DNA structures apart during cell division and resupplying nerve junctions (the synapse) with neurotransmitters (such as seratonin). The precise mechanisms by which these elaborate molecular machines, which are composed of tens of thousands of exquisitely arranged atoms, are able to `walk' are complex and incompletely understood. To see how they work, it is necessary to visualize these complex structures in three dimensions at sufficient levels of detail to resolve individual atoms? and to follow molecular rearrangements that happen while the motors step forward. This goal, however, has long remained out of reach due to the extreme technical challenges involved. We have addressed this problem by developing new methods to analyze images of frozen motor-filament assemblies collected by latest-generation electron microscopes. This approach, known as cryo-electron microscopy, allows us to directly visualize the three dimensional shape of individual molecular motor proteins attached to their partner filaments. During the previous funding period, we solved 3D structures of truncated single `feet' (one motor domain) of kinesin and myosin motors attached to their partner filaments, showing in atomic detail how these structures changed when molecules of ATP fuel were bound and consumed. We also captured a 3D structure of an intact pair of kinesin molecules (dimer) caught in mid- step on a microtubule. This allowed us to visualize, for the first time, a way in which the two `feet' of kinesin can pull on each other in a way to stay coordinated while walking. In our ongoing research we are improving our methods to capture more intermediates in the stepping process of kinesin, in order to gain a complete more understanding of how it walks. We are improving our analysis methods to better resolve precise chemical details within these structures. Finally, we are extending our approach to understand how a pair of myosin molecules can walk along the actin filament. Results of our studies are expected to aid the development of a new generation of pharmaceutical agents for treating cancer and a wide variety of other diseases.